Pathogenicity Assay for Cochliobolus heterostrophus G-Protein and MAPK Signaling Deficiency Strains

Ofir Degani

Tel-Hai College, Tel-Hai, Israel; Migal-Galilee Research Institute, Kiryat Shmona, Israel.

DOI: 10.4236/ajps.2014.59145                

Abstract

Cochliobolus heterostrophus is an agriculturally important and emerging model pathogen for studying the signaling hierarchies' role during the maize host colonization. In particular, G-protein and MAPK-linked pathways are playing a major role during pathogenesis. Although gene disruption studies are an efficient way of identifying the role of these cascades, differentiating between the mutant strains’ virulence ability may become an intricate task. For example, in C. heterostrophus, mutants in a G-protein α subunit gene, cga1, are defective in mating and appressorium formation, but unlike mutants in homologous genes in other fungal pathogens, the cga1 mutants remained highly virulent to corn under some host physiological conditions. Here, we used the cga1 strain as a model for developing an in vivo sensitive and accurate pathogenicity assay. A detailed and well controlled analysis of wild type (WT) and cga1 pathogenic behavior revealed that detached leaves are significantly more vulnerable to the disease than intact ones. In intact leaves, cga1 mutants were less infective of maize under most conditions. This difference was maximized when the first seedling leaf was chosen for inoculation and when the infected leaves, with spores or mycelia fragments droplets, were incubated for a period of four days. This optimal condition set enabled us to classify the C. heterostrophus G-protein signaling mutants deficient in α, β or both subunits in order of decreasing virulence: WT > cga1> cgb1> cga1 cgb1. The method presented proved to be accurate and sensitive enough to identify even slight variations in virulence. Moreover, it could be modified for use in studies of other foliar phytoparasitic fungi.

Keywords

Cochliobolus heterostrophus; G-Protein; Maize; MAPK; Signal Transduction; Virulence

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Kump, K.L., Bradbury, P.J., Wisser, R.J., Buckler, E.S., Belcher, A.R., Oropeza-Rosas, M.A., Zwonitzer, J.C., Kresovich, S., McMullen, M.D. and Ware, D. (2011) Genome-Wide Association Study of Quantitative Resistance to Southern Leaf Blight in the Maize Nested Association Mapping Population. Nature genetics, 43, 163-168. http://dx.doi.org/10.1038/ng.747.
[2]	Holley, R. and Goodman, M. (1989) New Sources of Resistance to Southern Corn Leaf Blight from Tropical Hybrid Maize Derivatives. Plant disease, 73, 562-564. http://dx.doi.org/10.1094/PD-73-0562
[3]	Smith, D., Hooker, A. and Lim, S. (1970) Physiologic Races of Helminthosporium maydis. Plant Disease Reporter, 54, 819-822.
[4]	Wei, J.-K., Liu, K.-M., Chen, J.-P., Luo, P.-C. and Stadelmann, O. (1988) Pathological and Physiological Identification of Race C of Bipolaris maydis in China. Phytopathology, 78, 550-554. http://dx.doi.org/10.1094/Phyto-78-550
[5]	Lee, J., Tattar, T., Berman, P. and Mount, M. (1992) A Rapid Method for Testing the Virulence of Cryphonectria parasitica Using Excised Bark and Wood of American Chestnut. Phytopathology, 82, 1454-1456. http://dx.doi.org/10.1094/Phyto-82-1454
[6]	Horwitz, B.A., Sharon, A., Lu, S.W., Ritter, V., Sandrock, T.M., Yoder, O.C. and Turgeon, B.G. (1999) A G Protein Alpha Subunit from Cochliobolus heterostrophus Involved in Mating and Appressorium Formation. Fungal Genetics and Biology, 26, 19-32. http://dx.doi.org/10.1006/fgbi.1998.1094
[7]	Ganem, S., Lu, S.W., Lee, B.N., Chou, D.Y., Hadar, R., Turgeon, B.G. and Horwitz, B.A. (2004) G-Protein Beta Subunit of Cochliobolus heterostrophus Involved in Virulence, Asexual and Sexual Reproductive Ability, and Morphogenesis. Eukaryotic Cell, 3, 1653-1663. http://dx.doi.org/10.1128/EC.3.6.1653-1663.2004
[8]	Lev, S. and Horwitz, B.A. (2003) A Mitogen-Activated Protein Kinase Pathway Modulates the Expression of Two Cellulase Genes in Cochliobolus heterostrophus during Plant Infection. Plant Cell, 15, 835-844. http://dx.doi.org/10.1105/tpc.010546
[9]	Lev, S., Sharon, A., Hadar, R., Ma, H. and Horwitz, B.A. (1999) A Mitogen-Activated Protein Kinase of the Corn Leaf Pathogen Cochliobolus heterostrophus is Involved in Conidiation, Appressorium Formation, and Pathogenicity: Diverse Roles for Mitogen-Activated Protein Kinase Homologs in Foliar Pathogens. Proceedings of the National Academy of Sciences of the United States of America, 96, 13542-13547. http://dx.doi.org/10.1073/pnas.96.23.13542
[10]	Degani, O. (2013) Cochliobolus heterostrophus G-Protein Alpha and Beta Subunit Double Mutant Reveals Shared and Distinct Roles in Development and Virulence. Physiological and Molecular Plant Pathology, 82, 35-45. http://dx.doi.org/10.1016/j.pmpp.2013.01.004
[11]	Hoffman, C.S. (2007) Propping up Our Knowledge of G Protein Signaling Pathways: Diverse Functions of Putative Noncanonical G Beta Subunits in Fungi. Science Signaling, 2007, pe3. http://dx.doi.org/10.1126/stke.3702007pe3
[12]	Gronover, C.S., Kasulke, D., Tudzynski, P. and Tudzynski, B. (2001) The Role of G Protein Alpha Subunits in the Infection Process of the Gray Mold Fungus Botrytis cinerea. Molecular Plant-Microbe Interactions, 14, 1293-1302. http://dx.doi.org/10.1094/MPMI.2001.14.11.1293
[13]	Liu, S. and Dean, R.A. (1997) G Protein Alpha Subunit Genes Control Growth, Development, and Pathogenicity of Magnaporthe grisea. Molecular Plant-Microbe Interactions, 10, 1075-1086. http://dx.doi.org/10.1094/MPMI.1997.10.9.1075
[14]	Degani, O. (2013) Construction of a Constitutively Activated Gα Mutant in the Maize Pathogen Cochliobolus heterostrophus. American Journal of Plant Sciences, 4, 2394-2399. http://dx.doi.org/10.4236/ajps.2013.412296
[15]	Degani, O., Maor, R., Hadar, R., Sharon, A. and Horwitz, B.A. (2004) Host Physiology and Pathogenic Variation of Cochliobolus heterostrophus Strains with Mutations in the G Protein Alpha Subunit, CGA1. Applied and Environmental Microbiology, 70, 5005-5009. http://dx.doi.org/10.1128/AEM.70.8.5005-5009.2004
[16]	Degani, O., Lev, S. and Ronen, M. (2013) Hydrophobin Gene Expression in the Maize Pathogen Cochliobolus heterostrophus. Physiological and Molecular Plant Pathology, 83, 25-34. http://dx.doi.org/10.1016/j.pmpp.2013.03.003
[17]	Turner, G. and Borkovich, K. (1993) Identification of a G Protein Alpha Subunit from Neurospora crassa That is a Member of the Gi Family. Journal of Biological Chemistry, 268, 14805-14811.
[18]	Gao, S., Choi, G.H., Shain, L. and Nuss, D.L. (1996) Cloning and Targeted Disruption of enpg-1, Encoding the Major in Vitro Extracellular Endopolygalacturonase of the Chestnut Blight Fungus, Cryphonectria parasitica. Applied and Environmental Microbiology, 62, 1984-1990.
[19]	Turgeon, B.G., Garber, R.C. and Yoder, O.C. (1985) Transformation of the Fungal Maize Pathogen Cochliobolus heterostrophus Using the Aspergillus nidulans amdS Gene. Molecular and General Genetics, 201, 450-453. http://dx.doi.org/10.1007/BF00331338
[20]	Segers, G.C. and Nuss, D.L. (2003) Constitutively Activated G[alpha] Negatively Regulates Virulence, Reproduction and Hydrophobin Gene Expression in the Chestnut Blight Fungus Cryphonectria parasitica. Fungal Genetics and Biology, 38, 198-208. http://dx.doi.org/10.1016/S1087-1845(02)00534-0
[21]	Noodén, L.D., Guiamét, J.J. and John, I. (1997) Senescence Mechanisms. Physiologia Plantarum, 101, 746-753. http://dx.doi.org/10.1111/j.1399-3054.1997.tb01059.x
[22]	Sinha, S. (1971) The Microflora on Leaves of Capsicum annuum (L.). In: Preece, T.F. and Dickinson, C.H., Eds., Ecology of Leaf Surface Microorganisms, Academic Press, London, 175-189.
[23]	Degani, O. (2014) G protein and MAPK Signaling Pathways Control the Ability of Cochliobolus heterostrophus to Exploit Different Carbon Sources. Advances in Biological Chemistry, 4, 40-50. http://dx.doi.org/10.4236/abc.2014.41007
[24]	Liu, G., Kennedy, R., Greenshields, D.L., Peng, G., Forseille, L., Selvaraj, G. and Wei, Y. (2007) Detached and Attached Arabidopsis Leaf Assays Reveal Distinctive Defense Responses against Hemibiotrophic colletotrichum spp. Molecular Plant-Microbe Interactions, 20, 1308-1319. http://dx.doi.org/10.1094/MPMI-20-10-1308
[25]	Gan, S. and Amasino, R.M. (1997) Making Sense of Senescence (Molecular Genetic Regulation and Manipulation of Leaf Senescence). Plant Physiology, 113, 313-319.
[26]	Kolattukudy, P.E., Rogers, L.M., Li, D., Hwang, C.S. and Flaishman, M.A. (1995) Surface Signaling in Pathogenesis. Proceedings of the National Academy of Sciences of the United States of America, 92, 4080-4087. http://dx.doi.org/10.1073/pnas.92.10.4080
[27]	Gilbert, R.D., Johnson, A.M. and Dean, R.A. (1996) Chemical Signals Responsible for Appressorium Formation in the Rice Blast Fungus Magnaporthe grisea. Physiological and Molecular Plant Pathology, 48, 335-346. http://dx.doi.org/10.1006/pmpp.1996.0027
[28]	Staples, R.C. and Hoch, H.C. (1995) Physical and Chemical Cues for Spore Germination and Appressorium Formation by Fungal Pathogens. In: Carroll, G.C. and Tudzynski, P. Eds., The Mycota, Springer-Verlag, Berlin/Heidelberg, 27-40.
[29]	Agrios, G.N. (2005) Plant Pathology. Academic Press Inc. (London) Ltd.